农杆菌介导瞬时沉默小麦HCP1的研究

doi:10.3864/j.issn.0578-1752.2015.20.018

Abstract

Abstract: 【Objective】Through transient silencing of the wheat HCP1 gene mediated by Agrobacterium, a simple, rapid, high efficient and reliable transgenic technique in wheat to identify the gene functions can be developed. The role of HCP1 gene in wheat in scavenging H₂O₂ that induced by inoculating Puccinia triticina was investigated using the transgenic plants overexpressing this target gene. The purpose of this study was to provide a theoretical basis for further understanding the signal transduction mechanism initiated by Puccinia triticina in wheat. 【Method】The Agrobacterium cells carrying the peGFP vector were cocultured into the wheat leaves, the explants for wheat transformation. The intensity and distribution of the fluorescence signal in leaves mediated by Agrobacterium strains of EHA105, LBA4404 and C58C1 were detected and the expressions of exogenous gene in leaves were examined. The transient silencing efficiency of HCP1 mediated by RNAi in the transgenic plants was detected by semi-quantitative PCR and real-time quantitative PCR as well as the Rohringer staining analysis. RT-PCR and qRT-PCR analyses were also performed to determine the expression of HCP1 after 12 h inoculation in strain C58C1, and after 72-96 h coculture in strains EHA105 and LBA4404. The changes of H₂O₂ stained by DAB were observed and the function of HCP1 was investigated in the transgenic leaves with transient silencing of the HCP1 gene inoculated race 260, an incompatible P. triticina strain.【Result】The GFP fluorescence signals were strongly detected at 48 h after inoculation of Agrobacterium strains with the explants. Three Agrobacterium strains all efficiently mediated the HCP1-RNAi transformation, with the strongest silencing efficiency by strain C58C1, followed by strains EHA105 and LBA4404. Rohringer staining suggested that strain EHA105 had a high efficiency to mediate the RNAi effects of HCP1, indicating that this strain had a high toxicity to cause the defensive reaction in cells. By contrast, strain C58C1 had higher silencing efficiency and less toxicity than strain EHA105, indicating that C58C1 could be used as the Agrobacterium strain to mediate wheat transformation. DAB staining revealed that H₂O₂ accumulated largely in wheat leaves with the transient silencing HCP1, showing comparable rate with the inoculation progress of P. triticina race 260. Thus, the accumulation of H₂O₂ was shown more amount in the transgenic leaves than the control group at times of 24 h and 48 h after inoculation of the leaf rust race.【Conclusion】Strain C58C1 inoculated with the binary plasmids for 72 h could transform wheat leaves with high efficiency. Transgenic analysis confirmed that HCP1 gene is involved in scavenging H₂O₂ when wheat inoculated with P. triticina strain.

Key words: wheat ( Triticum aestivum L.), transient silencing, HCP1, Puccinia triticina , H₂O₂

LIU Peng, WANG Shuai-shuai, YANG Gao-shan, ZHANG Heng, HAN Sheng-fang, WANG Dong-mei. Transient Silencing of HCP1 Gene in Wheat Mediated by Agrobacterium[J].Scientia Agricultura Sinica, 2015, 48(20): 4188-4196.

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